Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium‐dependent protein kinase 3

Abstract

Summary Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium‐dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3‐dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5‐3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5‐3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non‐phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5‐3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

Profiling of myristoylation in Toxoplasma gondii reveals an N-myristoylated protein important for host cell penetration

“…To generate pGra_5’UTRTUB_MIC7_HA, the Tub 5’UTR was amplified from gDNA using primer pair 37/38, and recodonised Mic7 sequence was amplified from pG140_MIC7_HA_iKO_loxP100 using primers 39 and 33. In parallel, the vector pGra_ApiAT5-3_HA ( Wallbank et al, 2019 ) sequence was amplified by inverse PCR using primer pair 34/40. The three resulting PCR amplicons were Gibson assembled to generate pGra_5’UTRTUB_MIC7 _HA.…”

“…Primer 41 was used to introduce the point mutations G2K and G3A into the Mic7 recodonised sequence. In parallel, the vector pGra_ApiAT5-3_HA ( Wallbank et al, 2019 ) was amplified by inverse PCR using primer pair 34/40. The three resulting PCR amplicons were Gibson assembled to generate pGra_5’UTRTUB_MIC7(G2K/G3A) _HA.…”

Signaling Cascades Governing Entry into and Exit from Host Cells by Toxoplasma gondii

“…This led the authors to conclude that CDPK3 might be important to amplify the response to a Ca 2+ stimulus, rather than being a binary switch from intracellular to extracellular parasites. CDPK3 has also been shown to be implicated in ion homeostasis (128) and metabolism (132) and might be implicated in controlling broader aspects of T. gondii cell biology, preparing the cell for intracellular or extracellular conditions. Like PKG, CDPK3 is dually acylated, and its association with the pellicle is essential for its function (51, 87, 91).…”

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

“…To generate pGra_5'UTRTUB_MIC7 _HA, the Tub 5'UTR was amplified from gDNA using primer pair 60/61, and recodonised Mic7 sequence was amplified from pG140_MIC7_HA_iKO_loxP100 using primers 62 and 56. In parallel, the vector pGra_ApiAT5-3_HA (Wallbank et al, 2019) sequence was amplified by inverse PCR using primer pair 57/63. The 3 resulting PCR amplicons were Gibson assembled to generate pGra_5'UTRTUB_MIC7 _HA.…”

“…Primer 64 was used to introduce the point mutations G2K and G3A into the Mic7 recodonised sequence. In parallel, the vector pGra_ApiAT5-3_HA (Wallbank et al, 2019) was amplified by inverse PCR using primer pair 57/63. The 3 resulting PCR amplicons were Gibson assembled to generate pGra_5'UTRTUB_MIC7(G2K/G3A) _HA.…”